A number of nucleoside analogs such as cytarabine, fludarabine, cladribine, capecitabine, gemcitabine and pentostatin are used clinically as highly effective anti-neoplastic agents. Among these, gemcitabine (2′,2′-difluoro-2′-deoxycytidine, Gemzar™ is of particular interest due to its unique activity against solid tumors and is presently used therapeutically to treat bladder, breast, lung, ovarian and pancreatic cancer.
Several self-potentiating mechanisms unique to this nucleoside analog are believed responsible for the activity of gemcitabine against solid tumors. The diphosphate metabolite of gemcitabine inhibits ribonucleotide reductase, which results in lower concentrations of intracellular deoxycytidine triphosphate (dCTP) and thus, increased incorporation of the triphosphate gemcitabine metabolite into DNA, which results in inhibition of DNA synthesis and blocks completion of the cell division cycle. Additionally, reduction in dCTP concentration up regulates the enzyme cytidine kinase, which is responsible for initial phosphorylation of gemcitabine, a necessary step in the inhibition of DNA synthesis by the drug. Finally, the triphosphate metabolite of gemcitabine is an inhibitor of cytidine deaminase, which is responsible for gemcitabine inactivation by conversion to the uridine metabolite. Accordingly, the additive nature of the above factors may explain the efficacy of gemcitabine in treating solid tumors.
Due to the lipophilic nature of the ProTides, these molecules can deliver nucleoside monophosphates directly in to the intact tumor cell. Previous studies have characterized multiple cellular transport mechanisms for nucleoside analog drugs and their derivatives (for a review, see Balimane et al., Adv. Drug Delivery Rev. 1999, 39, 183-209). A relatively hydrophilic compound, gemcitabine has limited ability to permeate plasma membranes via passive diffusion and several studies have demonstrated that gemcitabine is a substrate for equilibrative and concentrative nucleoside transporters (ENT's and CNT's respectively). Specifically, gemcitabine is transported by human ENT1, ENT2, CNT1 and CNT3, but not the purine-selective concentrative transporter CNT2 (see Mackey et al., Cancer Res. 1998, 58, 4349-4357; Mackey et al., J. Natl. Cancer Inst. 1999, 91, 1876-1881; and Fang et al., Biochem. J. 1996, 317, 457465).
U.S. Pat. No 4,808,614 discloses 2,2′-difluoronucleosides which are known anti-viral and anti-tumor agents, in particular 1-(2-oxo-4-amino-1H-pyrimidin-1 yl)-2-desoxy-2,2′-difluororibose (commonly known as Gemcitabine).
J. Org. Chem. Vol. 64, No. 22, 1999 disclosed a process for the selective protection of 4-NH2, 3′-OH and 5′OH positions of gemcitabine either as monoprotected or diprotected and were synthesized in good yield by employing commonly used di-tert-butyl dicarbonate. However, this publication does not provide preparation of phosphoramidate derivatives of gemcitabine and is esentially directed to PBR ligands of gemcitabine such as those having isoquinoline moiety.
U.S. Pat. No 7,951,787 discloses phosphoramidate derivatives of nucleotides such as 2′-deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzoxy-L-alaninyl)] phosphate (also referred to as gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate of Formula I). Methods for chemically synthesizing these derivatives are disclosed in this patent by reacting gemcitabine or its structural variants with appropriate phosphochloridate in the presence of N-methylimidazole followed by purification of the product by column chromatography, eluting with dichloromethane/methanol 95:5 to give pure product as a white foamy solid with very low yield of 16%.

The purity and the yield of gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate derivative as per the '787 patent are not satisfactory as starting nucleotide used in this reaction has two polar functional groups (3′-hydroxy and 4-amino), which can also form phosphoramidate ProTide esters along with 5′-hydroxy group, which is required for the formation of gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate of Formula I. Also the said process involves the column chromatographic purification for isolating the desired compounds; such processes are tedious, labor intensive and time consuming and hence not viable for commercial scale operations. The above described process is schematically represented as follows:

To overcome the diffuculties associated with the '787 patent, the present inventors have tried an improved process for preparing gemcitabine phosphoramidate ProTides such as gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate, wherein only 3′-hydroxy protected gemcitabine is used as starting material for coupling with ProTide intermediate using either N-methyl imidazole (NMI) or t-butyl magnesium halide (Br or Cl) followed by deprotection and column chromatographic purification which resulted gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate in an overall yield of 35-65%. This process resulted in a chemical purity of greater than 95% as a mixture of diastereomers and mixture of both diastereomers in approximately 2:1 ratio having around 1% of methoxy impurities. A similar process is described in Slusarczyk et al; J. Med. Chem.; 2014, 57, 1531-1542.

It is therefore an object of the present invention to provide a process for the preparation of gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate) in high yield and purity. Improvements include protection of both polar functional groups (3′-hydroxy and 4-amino) of gemcitabine followed by coupling with ProTide intermediate and final deprotection to obtain gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate with higher overall yield and purity of about 80-90%; it is evident that the new process represents a valuable alternative to the direct coupling of mono-protected gemcitabine with ProTide (35-65% yield).